Gag n gift

ABSTRACT

Assembly of the HIV-1 virus involves, in part, strong interactions between the capsid (CA) domains of the Gag polyprotein. During maturation, the core of HIV-1 virions undergoes profound morphological changes due primarily to proteolysis of the CA domain from other Gag domains which may allow for more efficient disassembly of the viral core in the early stages of infection. The host protein cyclophilin A (CypA), a cis-trans prolyl isomerase, in some way seems to assist in this assembly/disassembly process. Using an unproteolyzed construct of CA, we show that binding of CypA induces a large-scale conformational change in CA that is independent of its cis-trans prolyl isomerase activity. This change appears to be mediated by Cys-198 of CA since mutation to Ala renders CypA unable to induce this change and alters the kinetics and stability of protein cores that may ultimately result in inefficient disassembly of viral cores. Alternately, mutation of the second CA Cys (C218A) allows for CypA-induced conformational changes but alters the kinetics and morphology of the protein cores that may ultimately result in inefficient assembly of viral cores. These studies show the importance of the CA Cys residues in mediating the contacts needed for viral assembly and disassembly.

INTRODUCTION

Assembly of human immunodeficiency virus type 1 (HIV-1) in host cells involves the oligomerization of the structural precursor proteins, Pr55^sup Gag^ and Pr160^sup Gag-Pol^, the binding of genomic RNA, the association of the complex with viral envelope glycoproteins on the plasma membrane, and budding and release of the assembled virus particle (Scarlata and Carter, 2003). The Pr55^sup Gag^ precursor is sufficient by itself to direct membrane-binding, association with RNA, budding, and release. Expression of Gag or some of its constituent domains results in formation of particles that are morphologically very similar to the native immature virus (Gross et al., 1998; Campbell and Rein, 1999; Ehrlich et al., 2001). Gag oligomerization is mediated through several points in its matrix (MA), capsid (CA), and nucleocapsid (NC) domains (von Poblotzki et al., 1993; Mammano et al., 1994; Recin et al., 1995; Dorfman et al., 1997). Subsequent to assembly and release of the virion from host cells, Gag is proteolytically cleaved by the protease (PR) in Gag-Pol into the MA, CA, NC, and p6 products. This processing results in profound morphological rearrangements that change the spherical immature particle to a structure with a conical core composed entirely of the CA protein. The structural basis for this change is proposed to mechanistically result from cleavage at the N-terminus of the CA domain, releasing a Pro residue (Pro1) which subsequently forms a salt bridge with residue Asp-51 creating an intramolecular loop (Gamble et al., 1996; Gitti et al., 1996; Gross et al., 1998; von Schwedler et al., 1998). The mechanism through which this process alters CA-CA contacts, which ultimately result in a change in virus morphology, is not clear. It is clear that the strong CA interactions that occur in the context of Gag must be significantly weakened to allow for disassembly of the virus upon entry into host cells in the early stages of infection.

Besides providing structural integrity to HIV-1 virions, the CA domain in Gag is critical for the incorporation of the host protein cyclophilin A (CypA) into the virus. CypA is a cis-trans prolyl isomerase and in some types of cells, HIV-1 requires CypA for full infectivity, but other closely related viruses, such as SIV, do not (Braaten et al., 1996). CypA binds to a site in the CA domain, and interestingly, the requirement for cyclophilin can be transferred to other viruses by inserting the CypA-binding site into their CA domains (Bukovsky et al., 1997). The function of CypA in the HIV-1 life cycle is unclear. It has been suggested that CypA serves as a point of first contact between the virion and T cells that possess cyclophilin receptors on their membrane (Sherry et al., 1998; Saphire et al., 1999). However, other studies suggest a role for cyclophilin in post-entry events (Steinkasserer et al., 1995; Braaten et al., 1996; Ackerson et al., 1998). Under some conditions, CypA has been shown to protect HIV-1 from dominant host restriction factors that block infection by targeting incoming viral CA. In other cases CypA appears to be essential for host restriction (Towers et al., 2003). CypA also appears to be necessary for the functional expression of viral protein R (Vpr) (Zander et al., 2003). Vpr participates in nuclear localization of the viral preintegration complex and induces G^sub 2^ cell-cycle arrest in infected, proliferating-T cells (Bukinsky and Adzhubei, 1999; Henklein et al., 2000). Also, since CypA is a cis-trans prolyl isomerase, it may affect viral assembly/dissembly through conformation changes that perturb diminish strong CA-CA interactions.

CypA binds to residues 87-92 in the HIV-1 CA domain of Gag (i.e., HAGPIA). The CA protein consists of two distinct predominantly α-helical domains linked by a short, unstructured region (Berthet-Colominas et al., 1999). The N-terminal domain, which is connected to the MA protein in the context of Gag, contains the CypA-binding site (Franke et al., 1994; Liang et al., 2003). The C-terminal domain, which is connected to the NC domain and p6 region in the context of Gag, plays an essential role in CA-CA interactions (Colgan et al., 1996; Kattendbeck et al., 1997; Lanman et al., 2002). Previous studies have shown that the binding of CypA to this site alters the structure in distal regions of the protein (Bosco et al., 2002), and we found that binding of CypA to the N-terminus of a immature form of capsid resulted in pronounced changes in the environment of a fluorescent probe covalently linked to one of the two Cys residues in the C-terminal domain (BonHomme et al., 2003).

In this study, we have used the changes in fluorescence of probes covalently attached to one of the two CA Cys residues as a read-out of the ability of CypA to modulate conformational changes in aminohexahistidine-tagged CA (His^sub 6^-CA), which should mimic the immature form of the protein. To better understand the mechanism underlying the changes induced by CypA-binding, we mutated each of the Cys residues into Ala or Ser and studied the behavior of these mutants. The CA Cys residues lie close to each other, but do not form a disulfide linkage, as indicated by their accessibility to Cys-interactive probes (Colgan et al., 1996; McDermott et al., 1996). Single Cys mutants of capsid have been previously studied. Cellular studies have shown that mutation of Cys-198 interferes with viral particle disassembly, whereas mutation of Cys-218 blocks virus assembly (McDermott et al., 1996). Our studies show that CypA-binding induces conformational changes in the C-terminal domain of His^sub 6^-CA oligomers which were mediated through Cys-198. CypA-binding may thus facilitate the transition of the virus to a mature, disassembly-competent form.

MATERIALS AND METHODS

Constructs

The HIV-1 CA sequence (derived from pBH10, GenBank accession number M15654) was subcloned into the pB6 vector, a derivative of pET 11d (Novagen, Madison, WI) that expresses aminohexahistidine-tagged proteins. Mutants of aminohexahistidine-tagged CA protein, abbreviated His^sub 6^-CA, were made using Gene Editor In Vitro Site-Directed Mutagenesis System (Promega, Madison, WI). Insert regions were confirmed by DNA sequencing. Glutathione S-transferase-fused CypA, wild-type, and H54Q, was a gift from Dr. D. Braaten and Dr. J. Luban from the Dept. of Microbiology. College of Physicians and Surgeons of Columbia University, New York. Two antibodies were used in this study. The first, NEN-NEA 9306 (NEN-DuPont, DuPont, Wilmington, DE), is a monoclonal antibody that we found to binds to the region that encompasses amino acids 75-98 in the CA domain as shown in Fig. 1 a. The second is a monospecific antibody raised against a peptide whose sequence was residues 151-160 in the CA C-terminal domain shown in Fig. 1 a (International Enzymes, Fallbrook, CA). The amount of antibody used in this study was based on the supplier's recommendation and was adjusted to give similar potencies.

Recombinant protein expression and purification

His^sub 6^-CA protein was expressed in Escherichia coli-strain BL21 (DE3) (Novagen) and purified by gravity chromatography using Ni-NTA Superflow agarose (Qiagen, Valencia, CA). His^sub 6^-CA was eluted with 250 mM imidazole and the protein was found to be >95% pure by 15%-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining. His^sub 6^-CA was dialyzed into 30 mM MES (2-(N-morpholino)ethanesulfonic acid) pH 6, 1 mM EDTA, 1 mM DTT and 20% glycerol, and stored at -70°C.